P598: EBI3/IL-27 DEPLETED MICROENVIRONMENT FAVOURS TUMOUR PROGRESSION IN CHRONIC LYMPHOCYTIC LEUKAEMIA
Authors: I. C. Botana, G. Pagano, W. Marina, P. M. Roessner, B. Qu, A. Ramsay, B. Stamatopoulos, M. Seiffert, J. Paggetti, E. Moussay
Journal: HemaSphere (2022)

Background: Chronic Lymphocytic Leukaemia (CLL) is the most common adult leukaemia in western countries. Unfortunately, CLL remains an incurable disease, evidencing the urgency to develop novel effective treatments for this malignancy. CLL cells are known to be highly dependent on interactions with non-malignant cells in their tumour microenvironment (TME) for survival and proliferation. Such dependency highlights the potential of immunotherapy as a therapeutic approach in CLL. Recently, several pro-inflammatory cytokines have emerged as a powerful tool to reactivate the immune system in a wide range of malignancies. IL-27 is a member of the IL-12 family of heterodimeric cytokines reported to have pleiotropic functions during cancer development in different malignancies. Based on the existing literature, we wondered whether IL-27 affects the development and progression of CLL. Aims: The goal of this study is to elucidate the role of IL-27 in CLL development and progression. Methods: We used a constitutive knockout mouse model of the EBI3 subunit of IL-27 to abrogate IL-27 expression in vivo. We performed adoptive transfer (AT) of TCL1 derived CLL cells into Ebi3-/- and WT mice, as well as generated the transgenic mouse model Eµ-TCL1-Ebi3-/-.The splenic TME of these mice was characterized using flow cytometry. In addition, in vivo neutralization of IL-27 using a blocking antibody was conducted. Immunodeficient Rag2-/- mice were also injected with TCL1-derived CLL cells and either WT or Ebi3-/- CD3+ T cells to assess the role of T-cells in leukaemia control in the presence and absence of IL-27. Finally, the transcriptional differences among WT and Ebi3-/- T cells were assessed using bulk-RNA sequencing, and their impact on T cell cytotoxicity was investigated both in humans and mice using in vitro killing assays. The serum levels of IL-27 in CLL patients and sick mice were quantified via ELISA and compared to those of healthy controls. Results: A strikingly enhanced CLL development and survival reduction was observed when CLL was AT into Ebi3-/- mice, as well as in the transgenic Eµ-TCL1-Ebi3-/- mice when compared to controls (A). Consistently, flow cytometry analysis revealed an increasingly immunosuppressive splenic TME in the absence of EBI3, characterized by an enrichment of highly activated and immunosuppressive Tregs and a terminally exhausted CD8+ T cell subset. The aforementioned results were recapitulated in an IL-27 neutralization experiment (B). Using Rag2-/- mice, we demonstrated that T cells from EBI3-/- mice were less efficient in controlling CLL development (C). RNA sequencing revealed major changes in CD8+ T cells transcriptional program, in particular a decreased expression of crucial transporters (D). Finally, in vitro killing assays showed an increased cytotoxicity of both human and murine T cells in the presence of IL-27, while serum protein quantification showed a decrease in the levels of IL-27 in both CLL patients (E) and sick mice (F) when compared to their healthy counterparts. Image:Summary/Conclusion: Overall, our results demonstrate the antitumor role of IL-27 in CLL development and progression by promoting T-cell-mediated anti-tumor immunity, as well as establish this cytokine as a potential immunotherapeutic agent in CLL.